Neoceroplatus betaryiensis nov. sp. (Diptera: Keroplatidae) is the first record of a bioluminescent fungus-gnat in South America

Abstract

Blue shining fungus gnats (Diptera) had been long reported in the Waitomo caves of New Zealand (Arachnocampa luminosa Skuse), in stream banks of the American Appalachian Mountains (Orfelia fultoni Fisher) in 1939 and in true spore eating Eurasiatic Keroplatus Bosc species. This current report observes that similar blue light emitting gnat larvae also occur nearby the Betary river in the buffer zone of High Ribeira River State Park (PETAR) in the Atlantic Forest of Brazil, where the larvae were found when on fallen branches or trunks enveloped in their own secreted silk. The new species is named Neoceroplatus betaryiensis nov. sp. (Diptera: Keroplatidae: Keroplatinae: Keroplatini) based on a morphological analysis. Neoceroplatus betaryiensis nov. sp. larvae emit blue bioluminescence that can be seen from their last abdominal segment and from two photophores located laterally on the first thoracic segment. When touched, the larvae can actively stop its luminescence, which returns when it is no longer being agitated. The in vitro bioluminescence spectrum of N. betaryiensis nov. sp. peaks at 472 nm, and cross-reactivity of hot and cold extracts with the luciferin-luciferase from Orfelia fultoni indicate significant similarity in both enzyme and substrate of the two species, and that the bioluminescence system in the subfamily Keroplatinae is conserved.

Full text

1
SCIENTIFIC REPORTS | (2019) 9:11291 | https://doi.org/10.1038/s41598-019-47753-w
www.nature.com/scientificreports
Neoceroplatus betaryiensis nov. sp.
(Diptera: Keroplatidae) is the rst
record of a bioluminescent fungus-
gnat in South America
Rafaela L. Falaschi1, Danilo T. Amaral2, Isaias Santos3, Adão H. R. Domingos3,
Grant A. Johnson3, Ana G. S. Martins3, Imran B. Viroomal3, Sérgio L. Pompéia3,
Jeremy D. Mirza4,5, Anderson G. Oliveira
5, Etelvino J. H. Bechara6, Vadim R. Viviani2 &
Cassius V. Stevani
6
Blue shining fungus gnats (Diptera) had been long reported in the Waitomo caves of New Zealand
(Arachnocampa luminosa Skuse), in stream banks of the American Appalachian Mountains (Orfelia
fultoni Fisher) in 1939 and in true spore eating Eurasiatic Keroplatus Bosc species. This current report
observes that similar blue light emitting gnat larvae also occur nearby the Betary river in the buer
zone of High Ribeira River State Park (PETAR) in the Atlantic Forest of Brazil, where the larvae were
found when on fallen branches or trunks enveloped in their own secreted silk. The new species is
named Neoceroplatus betaryiensis nov. sp. (Diptera: Keroplatidae: Keroplatinae: Keroplatini) based
on a morphological analysis. Neoceroplatus betaryiensis nov. sp. larvae emit blue bioluminescence
that can be seen from their last abdominal segment and from two photophores located laterally on
the rst thoracic segment. When touched, the larvae can actively stop its luminescence, which returns
when it is no longer being agitated. The in vitro bioluminescence spectrum of N. betaryiensis nov. sp.
peaks at 472 nm, and cross-reactivity of hot and cold extracts with the luciferin-luciferase from Orfelia
fultoni indicate signicant similarity in both enzyme and substrate of the two species, and that the
bioluminescence system in the subfamily Keroplatinae is conserved.
e family Keroplatidae comprises of at least 92 genera and approximately 950 species1–5, distributed in all bio-
geographic regions. It is comprised of three subfamilies - Arachnocampinae, Macrocerinae, and Keroplatinae6,
however Papp and Ševčik proposed a new subfamily7 named Sciarokeroplatinae based on a single species found
the Oriental region. In the Neotropical region, there exist 32 genera and more than 200 species, of which 40 occur
in Brazil5,8. Keroplatids inhabit mainly moist tropical forests usually associated with fungi. Adults can be collected
in dark and humid places like stream banks and wet caves by sweeping in low vegetation, under hanging rocks
or decaying logs. Usually, ies are caught by Malaise traps, UV lamp traps and occasionally in yellow pan traps4.
Larvae are also present in moist and dark places, like caves, trunks, slits in stones, and have a variety of feeding
habits. Predaceous larvae are seen in all subfamilies9, whereas mycophagy characterizes Keroplatinae4,10.
A few articles report on the function of bioluminescence of Diptera11–13. e larvae of luminous species such
Archnocampa luminosa and Orfelia fultoni are carnivorous, however the Japanese Keroplatus nipponicus is fungi-
vorous. Cannibalism is also common in the case of O. fultoni. Webs can be found in luminous and non-luminous
1Departamento de Biologia Estrutural, Molecular e Genética, Programa de Pós-Graduação em Biologia Evolutiva,
Universidade Estadual de Ponta Grossa, Ponta Grossa, PR, Brazil. 2Depto Física, Química e Matemática, Graduate
School of Biotechnology and Environmental Monitoring (UFSCar), Sorocaba, SP, Brazil. 3IPBio - Instituto de Pesquisas
da Biodiversidade, Iporanga, SP, Brazil. 4Departamento de Química, Instituto de Ciências Ambientais, Químicas e
Farmacêuticas, Universidade Federal de São Paulo, Diadema, SP, Brazil. 5Departamento de Oceanografia Física,
Química e Geológica, Instituto Oceanográfico, Universidade de São Paulo, São Paulo, Brazil. 6Departamento de
Química Fundamental, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil. Correspondence and
requests for materials should be addressed to E.J.H.B. (email: ebechara@iq.usp.br) or V.R.V. (email: viviani@ufscar.br)
or C.V.S. (email: stevani@iq.usp.br)
Received: 1 March 2019
Accepted: 18 July 2019
Published: xx xx xxxx
OPEN
Content courtesy of Springer Nature, terms of use apply. Rights reserved

2
SCIENTIFIC REPORTS | (2019) 9:11291 | https://doi.org/10.1038/s41598-019-47753-w
www.nature.com/scientificreports
www.nature.com/scientificreports/
mycetophilids. ey are sticky and, in some cases, poisonous (i.e., contains oxalic acid as in the case of O. fultoni).
Bigger arthropods such as cockroaches and ants were found caught in the webs of O. fultoni14. Sivinski performed
experiments using transparent and blackened petri dishes covered with adhesive to verify whether light emission
by O. fultoni can attract insects11. Most insects captured consisted of small Diptera (cecidomyids and phorids),
which corroborated his hypothesis that preys were disoriented by the light emitted by the larva. In the case of a
fungivorous species, like K. nipponicus, Sivinski hypothesized bioluminescence can perform other functions such
as repelling negatively phototropic predators or as an aposematic signal12.
e subfamily Keroplatinae holds the highest number of genera (70) and species (677), in which it is possi-
ble to recognize two distinct tribes according to Matile2,15: Keroplatini and Orfeliini. e tribe Keroplatini con-
tains 153 species in 21 genera16, which are characterized mainly by short, two-segmented palpi and laterally
compressed or otherwise modied antennae. Of the few known immature specimens, the larva always has four
anal lobes. Bioluminescence in Diptera is reported only in the Keroplatidae family in the genera Arachnocampa
Edwards (Arachnocampinae), Keroplatus Bosc (Keroplatinae: Keroplatini), and Orfelia Costa (Keroplatinae:
Orfeliini)4,16,17. Light emission by a species of Mallochinus Edwards of the Keroplatini group is uncertain18. Yet,
according to Lloyd19 an unknown luminous species of Keroplatidae was found in New Guinea.
Notably, within the Keroplatidae there are at least two morphologically and biochemically distinct biolumi-
nescent systems, namely those of Arachnocampa and O. fultoni Fisher20, whilst the bioluminescent system of
Keroplatus species remains unknown. In Arachnocampa larvae, bioluminescence is produced by a lantern at
the tip of the abdomen which is derived from Malpighian tubules20, and involves an ATP-dependent luciferase
as in the case of beetle luciferases21. e chemical structure of the Arachnocampa luciferin was shown to derive
from xanthurenic acid and tyrosine but has not yet been elucidated22. On the other hand, the bioluminescence
system of O. fultoni, produced by translucent areas associated with rows of black bodies, involves an unknown
heterodimeric 140 kDa luciferase, an unknown luciferin and a Substrate Binding Fraction (SBF), which appar-
ently releases luciferin in the presence of reducing agents20. Notably, there is no cross-reaction between either the
luciferin or the luciferase of Arachnocampa and Orfelia.
e genus Neoceroplatus Edwards is represented by twelve species worldwide, of which only N. samiri Khalaf
is from the Neartic region. e other eleven species reportedly occur in the Neotropics4. Seven Neoceroplatus
species are known: N. hodeberti Matile, N. lauroi Lane, N. punctipes Matile, N. minimax Edwards, N. dissimilis
Matile, N. paicoenai Lane and N. spinosus Matile; the last four ones found in the State of São Paulo. Matile oers a
taxonomic key for all these Neotropical species2. In this work, a new species of Keroplatini is described occurring
in a conservation area of Atlantic Forest in São Paulo State named Betary Reserve, which is the rst record of
blue bioluminescent species of Diptera in the Neotropical region. Additionally, this work increases the number
of known Neoceroplatus species to thirteen. Importantly, its luciferin and luciferase seem to be similar to the ones
present in O. fultoni as attested by cross-reaction assays between both species.
Results and Discussion
Observation of larval behavior and bioluminescence. Larvae are usually found on fallen branches
or tree trunks, where they are lodged between the wood and surrounded by their secreted mucus. ey can also
be found on tree trunks about a meter above the ground (Fig.1). Typically, two or three larvae can share a single
branch, although as many as 15 have once been collected from a single branch. Some larvae were also spotted in
association with an unidentied species of brownish polypore mycelium. e larvae are very active, especially at
night and can move constantly whilst completely covered by mucus. When disturbed, they quickly move under
Figure 1. Dierent locations and habitats where larvae of Neoceroplatus betaryiensis nov. sp. were
photographed. (A) Decaying log where larvae were collected. (B) Larvae on the surface of the log surrounded by
a web-like mucus. (C) Association of a larva with a Favolus brasiliensis (Fr.) Fr. mushroom raised in a terrarium.
(D) Photo of a typically translucid N. betaryiensis sp. nov. (E) Details of the larva head and (F) last abdominal
segment.
Content courtesy of Springer Nature, terms of use apply. Rights reserved

3
SCIENTIFIC REPORTS | (2019) 9:11291 | https://doi.org/10.1038/s41598-019-47753-w
www.nature.com/scientificreports
www.nature.com/scientificreports/
their mucus. Before pupation, they construct a cocoon on the surface of the log beneath either moss or fungi,
where the pupae stay until the adult emerges (Fig.2). Pupae are also bioluminescent when observed using a CCD
camera (image not presented).
Neoceroplatus betaryiensis nov. sp. larvae emit blue light from their last abdominal segment and from two pho-
tophores located laterally on the rst thoracic segment (Fig.3). Until now, it was not possible to ascertain whether
the bioluminescence is associated to the black bodies as in the case Ofelia fultoni and possibly of Keroplatus spp.,
but with the presence of several dark granules spread along the body suggest this is a possibility. Light emission
is turned o when the larva is touched and begins again a few minutes aer this mechanical agitation ceases. We
were not able to observe the eating habits of the larvae, although the absence of remnants of insects trapped in the
web with the proximity of fungi suggest that the larvae could be fungivorous.
Figure 2. Life cycle of Neoceroplatus betaryiensis nov. sp. (A) Pupal stage. (B) Emerged adult female. (C)
Emerged adult male.
Figure 3. Bioluminescence of Neoceroplatus betaryiensis nov. sp. larvae. (A) Light emission under illumination
and (B) in the dark. (C) Detailed view of the two photophores located laterally on the rst thoracic segment.
Content courtesy of Springer Nature, terms of use apply. Rights reserved

4
SCIENTIFIC REPORTS | (2019) 9:11291 | https://doi.org/10.1038/s41598-019-47753-w
www.nature.com/scientificreports
www.nature.com/scientificreports/
Unexpectedly, a dierent larva was once pulled out from the underside of a fallen leaf, showing diuse blue
bioluminescence apparently consisting of small brown photophores along the whole body (Fig.4A,B). It exhib-
ited bizarre behavior compared to other larvae observed, as it moved slower and exposed itself more than was
previously observed. At rst, it was thought to be another luminous dipteran species. However, when kept in
a terrarium, the larva entered the pupal stage two weeks later and aer eleven days, an ichneumonid parasitic
wasp emerged from the pupa (Fig.4C). Luminous mycetophilids are reportedly attacked by hymenopterous par-
asitoids12 and this diuse bioluminescence could be the result of either a defensive reaction against the parasite
or the consequence of internal organ damage spreading the photogenic material along the body of the larva.
However, it may also belong to another new species that can emit light along the whole body as observed in
Keroplatus nipponicus13.
Chemiluminescence and luciferin-luciferase cross-reaction. Whole Neoceroplatus betaryiensis n ov.
sp. larvae were homogenized in the extraction buer. e resulting homogenate displayed considerable light
emission. Aer centrifugation a weaker luminescence remained in the supernatant, indicating that the lucif-
erin and luciferase are active and solubilized in these conditions. e supernatant or “cold extract” showed a
gradual increase in light emission to a higher intensity upon the addition of DTT (dithiothreitol) as a reducing
agent. is is similar to what occurs with crude extracts of Orfelia, consistent with the presence of the Substrate
Binding Fraction (SBF) which retains luciferin. Addition of O. fultoni hot extract that contains the luciferin to
Neoceroplatus cold extract (luciferase-rich fraction) also increased light emission. e spectrum obtained by
cross-reacting the cold extract of N. betaryiensis nov. sp. and the hot extract of O. fultoni in the presence of DTT
shows a maximum intensity of around 472 nm (Fig.5), which matches the recently reported value for O. fultoni
Figure 4. Parasitized unidentied luminous dipteran larva. (A) Larva under illumination and (B) light
emission from the entire body. (C) Ichneumonid parasitic wasp that emerged from the pupa.
Figure 5. Chemiluminescence spectrum obtained from the reaction of luciferase and hot extracts of Orfelia
fultoni (gray) and Neoceroplatus betaryiensis nov. sp. cold extract plus O. fultoni hot extract (black).
Content courtesy of Springer Nature, terms of use apply. Rights reserved

5
SCIENTIFIC REPORTS | (2019) 9:11291 | https://doi.org/10.1038/s41598-019-47753-w
www.nature.com/scientificreports
www.nature.com/scientificreports/
luciferin-luciferase system23. e addition of hot extract of N. betaryiensis nov. sp. to the puried luciferase of O.
fultoni also resulted in blue light emission. ese results indicate that N. betaryiensis nov. sp. and O. fultoni share
either very similar or identical luciferin substrates and luciferase enzymes.
Molecular phylogenetic studies. A molecular phylogenetic analysis was conducted with N. betaryiensis,
Orfelia fultoni and the non-luminescent Neoditomiya sp., using the mitochondrial gene of cytochrome oxidase
I (COI). COI barcode-gene is useful to recognize closely related species, to investigate their relative phyloge-
netic position, and to analyze if there was an evolutionary relationship among the luciferin-luciferase systems
of Keroplatinae. The molecular phylogenetic analysis showed that the fungus-gnat N. betaryiensis nov. sp.
is positioned as a sister-group of the luminescent Keroplatus genus (Fig.6), followed by the non-luminescent
Neoditomyia (Lane & Sturm) and the luminescent O. fultoni, which is in agreement with the literature2,10. e
placement of the non-bioluminescent Neoditomyia sp. as a sister-group of Keroplatinae subfamily was also
observed. Interestingly, Orfelia-type luciferin and its Substrate Binding Protein (SBF) has been recently reported
in Neoditomyia sp.23. Both the luciferin and SBF of Neoditomyia were able to cross-react with puried O. fultoni
luciferase, resulting in an emission spectrum that overlaps with that of O. fultoni extracts. e cross-reactions
between O. fultoni and N. betaryiensis nov. sp. strongly suggest that the same bioluminescent system is also
shared by these genera. Altogether, the COI gene analysis and biochemical results indicate that Neoditomyia spp.,
Neoceroplatus betaryiensis and Orfelia fultoni, and by inference Keroplatus species, share the same bioluminescent
system.
Taxonomical description. Neoceroplatus betaryiensis Falaschi, Johnson & Stevani nov. sp. (Figs1B to 4B,
S1A to S5D).
Material examined. Holotype: Male, BRAZIL, São Paulo, Iporanga, Reserva Betary, IPBio – Instituto de
Pesquisas da Biodiversidade, 24°35′27″S 48°37′44″W, 120 m, manual collection on the underside of a leaf (on
June 14th 2017 one larva was collected and on August 24th 2017 the adult male emerged), Domingos, A. H.
R., Santos, I. & Johnson, G. A. cols. [MZUSP- MZ052800] (specimen pinned with terminalia on permanent
slide). Paratypes: Two females, same data as holotype, except 01.v.2017 (larva collected) 24.v.2017 (adult female
emerged) [MZUSP-MZ052801] (specimen pinned), [MZUSP-MZ052802 (specimen on permanent slide); one
larva, same data as holotype, except 15.v.2017, [MZUSP-MZ052803] (in 80% ethanol); two larvae, same data
as holotype, except 16.iv.2017, [MZUSP-MZ052804] (in 80% ethanol), [MZUSP-MZ052805] (in permanent
slide); pupa exuvium, same data as holotype, except 24.viii.2017, [MZUSP-MZ052806] (in permanent slide); net
remains, same data as holotype, except 24.viii.2017, [MZUSP-MZ052807] (in 80% ethanol).
Etymology: e specic epithet refers to the Betary brook, in whose banks the specimens were collected.
Figure 6. Phylogenetic tree of Keroplatidae bioluminescent and non-bioluminescent species using
mitochondrial gene cytochrome oxidase I (COI). Species surrounded by a grey rectangle are bioluminescent.
Species whose name is displayed in pale blue share the same specic luciferin and SBF and the species with
name in dark blue, share the same specic luciferase and luciferin.
Content courtesy of Springer Nature, terms of use apply. Rights reserved

6
SCIENTIFIC REPORTS | (2019) 9:11291 | https://doi.org/10.1038/s41598-019-47753-w
www.nature.com/scientificreports
www.nature.com/scientificreports/
Diagnosis and comments. Neoceroplatus betaryiensis nov. sp. can be distinguished from the other
Neotropical Neoceroplatus¸ especially from N. dissimilis, its closest species, by the shape of the genitalia, par-
ticularly the gonostylus (FigsS3E, S4B,C) and the absence of spines in the gonostylus as appears in N. paicoenai.
Male. Body length (without antennae): 5.2 mm (Fig.2C). Wing length: 3.75 mm (Fig.S1F). Terminalia length:
0.28 mm (FigsS3A,C,E; S4A,B). Head: Brownish. ree ocelli in triangular position, each positioned on a callus.
e lateral ocellus larger than middle one (Fig.S1D). e distance between the lateral ocelli two times its own
diameter, separated from the edge of the eye at a distance slightly higher than its own diameter. Median ocellus
approximately 3/4 smaller than lateral ocellus. Compound eyes are large, about 1.5 times higher than wide in
lateral view, covered with microsetae. Antenna (Fig.S1A,B) three times higher than head, attened laterally,
yellowish-brown. Scape and pedicel cylindrical, both slightly wider than high, brownish. Flagellum expanded
and attened, with 14 agellomeres, dorsal and ventral macrosetae covering agellomeres. Terminal agellomere,
longer than wide, with a minute, thin yellow-whitish apical process at the apex, in a shallow notch (Fig.S1B).
Labrum and labella weakly sclerotized; labellum exceeding the ventral edge of the eyes. Palpus bi-segmented.
Second palpomere elongated, covered with setae laterally, pointed at the apex, as long as face and clypeus com-
bined length; medially membranous, weakly sclerotized. orax: Mostly yellowish, with brownish spots laterally.
Antepronotum brownish, covered with macrosetae; proepisternum brownish, bare; anterior spiracle surrounded
by setae; anepisternum bare, apically yellowish and brownish at the base and internal edge; katepisternum and
anepimerum bare, mostly yellowish, with a brownish spot proximally; laterotergite mostly brownish, covered
with long and sparse macrosetae. Scutum brownish, densely covered with macrosetae. Scutellum brownish, cov-
ered with long macrosetae. Mediotergite bare, mostly brownish. Halter with brownish knob and stem mostly
yellowish, with a row of setae. Wing: (Fig.S1F) Wing membrane infuscate brown to dark brown mainly on distal
half, basal cells yellowish with small brown maculae; the minor hyaline area between R4 and R5, and C to M1.
Microsetae irregularly scattered throughout wing. Veins brown to dark brown. Strong setation on C, R1, R4 and
R5. Humeral lightly shorter than half of R1. Vein C exceeding apex of R5, at a distance beyond R5 that is roughly 1/4
the distance between R5 and M1 apex. R4 ending at R1 apex, bending posteriorly with almost right angle with R5.
M1, M2, M4 and anal veins not reaching the wing margin. A1 as long as CuP vein. Legs: Mostly white to yellowish.
Fore coxa white-yellowish with brownish spot and covered with macrosetae on its all anterolateral face. Mid coxa
white-yellowish with two brownish spots covered with macrosetae anterolaterally; hind coxa white-yellowish with
a large brownish spot covered with macrosetae only laterally. Femora white-yellowish, covered with setae, with an
upper brownish spot antero-laterally. Tibiae and corresponding tarsi yellowish to pale brown, covered with regu-
lar rows of setae. Abdomen: Covered with macrosetae. Mostly yellowish ventrally and laterally, with large brown-
ish areas dorsally. Sternites 3–6 with two brownish marks in each lateral margin (Fig.S2A–C). Terminalia: Mostly
yellow (Fig.S3A,C,E) covered with long, dense macrosetae (Fig.S4A,B). Tergite 9 yellow-brownish, V-shaped,
longer than wide (FigsS3C,G, S4A). Cerci yellow, poorly sclerotized, covered with dark setae (FigsS3C,G, S4A).
Gonocoxites partially fused basally, yellowish, almost entirely covered with long and dark setae, and few short and
dark setae (Fig.S3A,E). Gonostylus dark brown, prominent, shark n-shaped, covered with long setae, and with
dark, thicker bristles on inner side (FigsS3E, S4B,C), with an interiorly-directed, antero-dorsal lobe (Fig.S3E).
Female. Body length (without antennae): 5.9 mm (Fig.2B). Wing length: 4 mm (Fig.S1G). Terminalia length:
0.39 mm (FigsS3B,D,F; S4D–E). As the male, except as follows. Head: Last agellomere slightly wider than long,
subquadrate (Fig.S1C). Wing: the minor hyaline area between R4 and R5, and C to M2 and the major hyaline area
reaching the apex of CuA. Humeral roughly more than half of R1. Vein C at a distance beyond R5 that is more than
one third the distance between R5 and M1. R4 divergent relative to R5 forming a 45°. Base of M4 weakly sclerotized.
A1 longer than CuP vein (Fig.S1G). Abdomen: (Fig.S2D,E) Dorsally yellowish with two large brownish spots on
tergites 2–7. Sternites 2–3 white-yellowish with one subtriangular brownish mark and rounded shaped on 4–5.
Legs: Mid coxa whitish with two dark-brownish spots covered with macrosetae antero-laterally, and a row with
four macrosetae at the lateral apex; hind coxa whitish with a large dark-brownish spot covered with macrosetae
laterally (Fig.S1E). Femora whitish, the hind femora with two brownish spots antero-laterally. Terminalia: mostly
brownish (FigsS3B,D,F; S4D,E) covered with long, dense macrosetae, without microsetae. Sternite 8 covered with
dense setae, inner margin straight; tergite 8 covered with setae; tergite 10 well developed; cercus formed by one
article, entirely covered with setae (Fig.S4D,E).
Immature. Body length: 20.1 mm (Figs1D, 3A, 4A and S5C); Pupa exuvium: 6,95 mm (Fig.S5D). Overall mor-
phology similar to other Keroplatini larvae. General color gray-whitish, head capsule brown, well sclerotized,
subquadrate (Fig.S5A,B). No distinctive modication of the body cuticle, except anterior portion of body divided
into four subquadrate areas, followed by narrow transverse lines towards posterior end (Figs1D–F, 3A, 4A and
S5C), giving a segmented texture to the integument. Cephalic capsule quadrangular, slightly longer than wide,
very short in relation to body width (Figs3C, S5A,B); a well-developed gena, ventral part of foramen magnum
longer than wide. Antenna very reduced, attened, ellipse-shaped, protruding above the antenna base, positioned
more dorsally. Labrum in continuity with the clypeal area. Premandible with row of elongated, exible teeth, sup-
ported by a pair of lateral chitinous arms. Mandible semicircular and bearing two rows of medially directed teeth;
Mandible well developed. Maxilla elongated, rather parallel distally, with een teeth on inner border. Cardo
slender, transverse (Fig.S5A). Secondary annulation on abdominal segments. Posterior end with a pair of lobose
triangular projections (Figs1D,F and 3A).
Comments. e anterior photophores are located on the rst thoracic segment of the larva and the posterior on
the last abdominal segments, without visible external morphological structures.
Content courtesy of Springer Nature, terms of use apply. Rights reserved

7
SCIENTIFIC REPORTS | (2019) 9:11291 | https://doi.org/10.1038/s41598-019-47753-w
www.nature.com/scientificreports
www.nature.com/scientificreports/
In the holotype and paratypes of Chetoneura shennonggongensis (Amorim & Niu) from the Zoology Museum
of University of São Paulo, Brazil24, there are no structures in the last segment similar to the “posterior papillae”,
described by Matile from the genera Arachnocampa, Keroplatus, and Macrocera in either C. shennonggongensis or
Neoceroplatus betaryiensis. ese structures can be properly described and understood from deeper histological
studies, which was not the aim of this study.
Conclusion
Here we report the discovery of the rst bioluminescent species of fungus-gnats of the family Keroplatidae in the
Neotropical region. Similar to the Palearctic and Oriental Keroplatus species, N. betaryiensis also lives under dead
logs and is probably sporophagous. e bioluminescence is blue, and likely shares the same luciferin-luciferase
system of the North-American Orfelia fultoni, and possibly of the Palearctic Keroplatus spp. ese ndings show
how Neotropical biodiversity is still poorly known25, despite being recognized as the most diverse biogeographic
region on the planet. Unfortunately, the anthropic pressure on natural areas has been increasing, causing distur-
bances in dierent habitats, with damage in megadiverse countries such as Brazil26. ese threats aect especially
small invertebrates, which have been extinguished at a much faster rate than their discovery and description27.
is reinforces the need for conservation policies for areas such as the Betary Reserve, a place that provides new
taxa for science, the Keroplatidae being one of them.
Methods
Diptera larva collection. e material presented here was collected by G.A.J., A.H.R.D. and I.S. on the
property of the non-governmental organization Instituto de Pesquisas da Biodiversidade (IPBio), municipality of
Iporanga, São Paulo State, Brazil. e Betary Reserve, the rst branch of IPBio, is located between the geograph-
ical coordinates 24°35′16″S; 48°37′44″W, a preserved area of ca. 60 hectares of dense ombrophylous forest in an
advanced regeneration status. e reserve is situated in the largest remaining continuous area of the Atlantic
Forest, and within the protective zone of the Touristic State Park of High Ribeira River (PETAR). e fungus
gnats were collected during a hot and rainy period, with relative humidity of 90%, and manually collected on
fallen trees.
On May 1st 2017, four larvae were collected and kept in terraria, and on May 24th two female adults emerged.
Another larva, with erratic behavior, collected on May 31, 2017 was parasitized by an ichneumonid wasp, which
emerged on June 13th. In the laboratory the larvae were kept in large glass terraria with a thin fabric cover. e
bottom of the terraria were covered with dried leaves, with branches leaning against the lateral glass walls, and
with thicker branches that harbored mushrooms of the species Favolus brasiliensis (Fr.) Fr. It was observed that
aer a few days the larvae migrated to the bottom of the mushrooms, where they built their web. Aer a few days
they went down to the leaves and about two weeks later they emerged as adults.
e female adults that emerged in the laboratory were primarily preserved in 70% ethanol. e larvae, which
were preserved in KAAD solution (100 mL kerosene, 700 mL of absolute alcohol, 100 mL of acetic acid and
100 mL of colorless house detergent), were collected in the eld. e larva remained in solution for 12 h and then
70% alcohol for 24 h. Aer that, the larva was removed and kept in 80% alcohol. Hot water prevented dryness and
rapid “wrinkling” and KAAD preserved color.
Larvae and adult preservation and microscopic examination. e material examined in this study
was deposited in the Diptera collection at the Museu de Zoologia da Universidade de São Paulo (MZUSP, São
Paulo, Brazil). e collected material was initially preserved in 80% ethanol. e terminalia were detached from
the abdomen, cleared in 10% KOH aqueous solution at 40 °C for 40–60 min and promptly washed rst in glacial
acetic acid and then in 80% ethanol for 15 min. Each specimen and wings were soaked for 15 min twice both in
absolute ethanol and then in xylenes (mixture of o,m,p-xylene) Synth, 98.5%. Aerwards, each specimen was
mounted in the permanent slide with Canadian balsam. Wing and terminalia were drawn aer mounting on
permanent slides. Photographs were taken with using a Leica DC camera either attached to a Leica MZ16 stere-
omicroscope or to a Leica DM2500 microscope. Stacking was performed with Helicon Focus 6 and edited with
the Adobe Photoshop CC 2017. General illustrations of male and female terminalia were drawn with the help of a
camera lucida attached to the microscope, vectorized using Adobe Illustrator CC 2017. e species was identied
using the available identication key present in Matile2 and by comparison with the types housed at the MZUSP
(with the closest species – Neoceroplatus dissimilis and the other with the type specimens available: N. dureti; N.
hodeberti; N. lauroi; N. monostylus; N. paicoenai and N. spinosus). e morphological terminology used here
follows the literature2,28.
Bioluminescence imaging. Imaging of bioluminescent larvae and pupae were done using a LightCapture
II CCD camera (ATTO, Tokyo, Japan).
Chemiluminescence assay. Between 3 to 6 entire Neoceroplatus betaryiensis nov. sp. larvae were homoge-
nized in 1 ml of Orfelia fultoni extraction buer (0.10 M phosphate buer, 1 mM EDTA, 1% Triton X-100, pH 7.0).
Hot-cold extract assays were performed using a previously established method for O. fultoni20,23. e homogenate
of the larvae was separated in two aliquots, one of which was centrifuged and the pellet discarded. e superna-
tant was le to react completely, with the remaining solution being termed the “cold extract”. e second aliquot
was treated with 10 mM DTT and was heated at 98 °C for 5 minutes in an anoxic atmosphere. is solution was
then centrifuged and the pellet discarded, leaving the “hot extract”. Puried O. fultoni luciferase and luciferin con-
taining hot extracts were prepared using larvae collected in North Carolina (USA) by one of the authors (VRV).
Light emission was measured in counts per second (cps) using an ATTO AB2200 luminometer (Tokyo, Japan).
Chemiluminescence spectra was recorded using an Atto LumiSpectra spectroluminometer (Tokyo, Japan).
Content courtesy of Springer Nature, terms of use apply. Rights reserved

8
SCIENTIFIC REPORTS | (2019) 9:11291 | https://doi.org/10.1038/s41598-019-47753-w
www.nature.com/scientificreports
www.nature.com/scientificreports/
Molecular phylogenetic analysis. e phylogenetic analysis of Keroplatidae species was performed using
the mitochondrial gene cytochrome oxidase I (COI). e nucleotide sequences were aligned using ClustalW algo-
rithm29 on MEGA 6.0 soware30. e jModelTest2 program31 was used to predict the best evolutionary model,
which resulted in GTR + G + I substitution model. e phylogenetic analysis used by the soware MrBayes 3.232,
through two separately runs with 10,000,000 generations each. e rst 25% of trees were discarded and concat-
enated to create a consensus tree.
References
1. Papavero, N. Family eroplatidae (Ceroplatidae, incl. Macroceridae). In A Catalogue of the Diptera of the Americas South of the
United States 19C, 1–22 (1978).
2. Matile, L. echerches sur la systematique et l’evolution des eroplatidae (Diptera, Mycetophilidae). Mem. Mus. Natn. Hist. Nat., ser
A, Zool 148, 1–682 (1990).
3. Evenhuis, N. L. Catalogue of the fossil ies of the world (Insecta: Diptera). (Bachuys Press, Leiden, 1994).
4. Evenhuis, N. L. Catalog of the eroplatidae of the World (Insecta: Diptera). Bishop Mus Bull Entomol 13, 1–178 (2006).
5. Oliveira, S. S., Falaschi, . L., Urso-Guimarães, M. V. & Amorim, D. S. Lista das espécies de Bibionomorpha (Diptera) do Estado do
Mato Grosso do Sul, Brasil. Iheringia Ser. Zool. 107, 1–8 (2017).
6. Matile, L. Description d’un eroplatidae du crétacé moyen et données morphologiques et taxinomiques sur les Mycetophiloidea
(Diptera). Ann. Soc. Entomol. Fr. 17, 99–123 (1981).
7. Papp, L. & Ševči, J. Sciaroeroplatinae, a new subfamily of eroplatidae (Diptera). Acta Zool. Hung. 51, 113–123 (2005).
8. Falaschi, . L. elações logenéticas entre os eroplatinae: posição de Orfeliini e relação entre seus gêneros (Diptera: eroplatidae).
(PhD esis, ibeirão Preto, Universidade de São Paulo, 2012).
9. Mansbridge, G. H. On the biology of some Ceroplatinae and Macrocerinae (Diptera, Mycetophilidae). Trans. . Entomol. Soc. Lond.
81, 75–92 (1933).
10. Matile, L. Phylogeny and evolution of the larval diet in the Sciaroidea (Diptera, Bibionomorpha) since the Mesozoic. In e Origin
and Biodiversity in Insects: Phylogenetic Tests of Evolutionary Scenarios (ed. Grandcolas, P.). Mem. Mus. Nat. Hist. Nat. A 173,
273–303 (1997).
11. Sivinsi, J. M. Prey attraction by luminous larvae of the fungus gnat Orfelia fultoni. Ecol. Entomol. 7, 443–446 (1982).
12. Sivinsi, J. M. Phototropism, bioluminescence and the Diptera. Fla. Entomol. 81, 282–292 (1998).
13. Osawa, ., Sasai, T. & Meyer-ochow, V. B. New observations on the biology of eroplatus nipponicus Oada 1938 (Diptera;
Mycetophiloidea; eroplatidae), a bioluminescent fungivorous insect. Entomol. Heute 26, 139–149 (2014).
14. Fulton, B. B. A luminous y larva with spider traits. Ann. Entom. Soc. Am. 34, 289–302 (1941).
15. Matile, L. Note sur les Macrocerini (stat. nov.) et description d’un genre et de sept espèces de la région éthiopienne (Diptera,
Mycetophilidae). Bull. Inst. Fond. Afr. Noire 34, 593–610 (1973).
16. Ševčí, J., Mantič, M. & Blagoderov, V. Two new genera of eroplatidae (Diptera), with an updated ey to the World genera of
eroplatini. Acta Entomol. Mus. Nat. Pragae 55, 387–399 (2015).
17. Baccetti, B., Crovetti, A. & Santini, L. Light-producing organs in eroplatus tipuloides Bosc and . reaumuri pentophthalmus Giglio-
Tos (Diptera: Mycetophilidae). Int. J. Morphol. Embryol. 16, 169–176 (1987).
18. Oba, Y., Branham, M. A. & Fuatsu, T. e terrestrial bioluminescent animals of Japan. Zool. Sci. 28, 771–789 (2011).
19. Lloyd, J. E. Insect Bioluminescence. In Bioluminescence in Action (ed. Herring, P. J.). Academic Press, New Yor, 241–272 (1978).
20. Viviani, V. ., Hastings, J. W. & Wilson, T. Two bioluminescent diptera: the North American Orfelia fultoni and the Australian
Arachnocampa ava. Similar niche, dierent bioluminescence systems. Photochem. Photobiol. 75, 22–27 (2002).
21. Trowell, S. C., Dacres, H., Dumancic, M. M., Leitch, V. & icards, . W. Molecular basis for the blue bio- luminescence of the
Australian glow-worm Arachnocampa richardsae, (Diptera: eroplatidae). Biochem. Biophys. es. Commun. 78, 533–539 (2016).
22. Watins, O. C., Sharpe, M. L., Perry, N. B. & rause, . L. New Zealand glowworm (Arachnocampa luminosa) bioluminescence is
produced by a rey-lie luciferase but an entirely new luciferin. Sci. ep. 8, 3278 (2018).
23. Viviani, V. ., Amaral, D. T., Bevilaqua, V. . & Falaschi, . L. Orfelia-type luciferin and its associated storage protein in the non-
luminescent cave worm Neoditomyia sp. (Diptera: eroplatidae) from the Atlantic rainforest: biological and evolutionary
implications. Photochem. Photobiol. 17, 1282 (2018).
24. Amorim, D. S., Niu, C. H., Li, X., Lei, C. H. & Clare, A. . Chetoneura shennonggongensis, a new species of cave-dwelling
eroplatini from China (Diptera: eroplatidae), with a discussion of the position of Chetoneura. Zootaxa 1716, 59–68 (2008).
25. Lewinsohn, T. & Prado, P. I. Quantas espécies há no Brasil? Megadiversidade 1, 36–42 (2005).
26. Carvalho, C. J. B. Padrões de endemismo e a conservação da biodiversidade. Megadiversidade 5, 77–86 (2011).
27. Carvalho, M. ., Bocmann, F. A., Amorim, D. S. & Brandão, C. . F. Systematics must embrace comparative biology and evolution,
not speed and automation. J. Evol. Biol. 35, 97–104 (2008).
28. Cumming, J. M. & Wood, D. M. Adult morphology and terminology. In Manual of Central American Diptera (eds Brown, B. V.,
Borent, A., Cumming, J. M., Wood, D. M., Woodley, N. E. & Zumbado, M. A.), NC esearch Press, Ottawa, 1, 9–50 (2009).
29. Higgins, D. et al. CLUSTAL W: improving the sensitivity of the progressive multiple sequence alignment through sequence
weighting, position-specic gap penal- ties and weight matrix choice. Nucleic Acids es. 22, 4673–4680 (1994).
30. Tamura, ., Stecher, G., Peterson, D., Filipsi, A. & umar, S. MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Mol.
Biol. Evol. 30, 2725–2729 (2013).
31. Darriba, D., Taboada, G. L., Doallo, . & Posada, D. jModelTest 2: more models, new heuristics and parallel computing. Nat.
Methods 9, 772 (2012).
32. onquist, F. et al. MrBayes 3.2: Ecient Bayesian Phylogenetic Inference and Model Choice Across a Large Model Space. Syst. Biol.
61, 539–542 (2010).
Acknowledgements
e authors are indebted to Dr. Carlos Lamas (MZUSP, Sao Paulo, Brazil), for access to the type material studied
in this work and Dr. Diego Fachin (USP, Ribeirão Preto, Brazil) who very kindly revised the description text of the
manuscript. We also thank Dr. Jay C. Dunlap (Dartmouth Geisel School of Medicine, USA) and Dr. Graham S.
Timmins (University of New Mexico, USA) for careful reading of the manuscript. is work was funded by grants
of Fundação de Amparo a Pesquisa do Estado de São Paulo (grants FAPESP 2010/05426-8 to VRV, 2013/16885-1
to CVS and 2017/22501-2 to AGO, EJHB and CVS), Brazilian National Research Council (CNPq/Universal
Project 401867/2016-1 to VRV and 306460/2016-5 to EJHB) and by the Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior - Brasil (CAPES - Finance Code 001) to RLF. is work was also partially supported
by funding from the Oce of Naval Research Global through grant ONR N62909-17-1-2103 to CVS and AGO.
Content courtesy of Springer Nature, terms of use apply. Rights reserved

9
SCIENTIFIC REPORTS | (2019) 9:11291 | https://doi.org/10.1038/s41598-019-47753-w
www.nature.com/scientificreports
www.nature.com/scientificreports/
Author Contributions
I.S., A.H.R.D., G.A.J., A.G.S.M., I.B.V. and S.L.P. discovered, collected and conducted experiments in the eld
with N. betaryiensis nov. sp. A.H.R.D. took all macro photographs, excepting the ones depicted in Figures 1D–F
and 4C, which were taken by G.A.J. R.L.F. was responsible for the identication, preparation, micrographies,
description and designation of the type-species and morphological comparative analysis with the other described
species of the studied genus. D.T.A. performed the molecular phylogenetic analysis. V.R.V. performed the cross-
reaction experiments, recorded luminescence spectra purified Orfelia fultoni luciferase and supervised the
biochemical and molecular aspects of bioluminescence. J.D.M., A.G.O., E.J.H.B. and C.V.S. planned, organized
and evaluated critically the experiments, and wrote the manuscript.
Additional Information
Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-47753-w.
Competing Interests: e authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional aliations.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International
License, which permits use, sharing, adaptation, distribution and reproduction in any medium or
format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre-
ative Commons license, and indicate if changes were made. e images or other third party material in this
article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the
material. If material is not included in the article’s Creative Commons license and your intended use is not per-
mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the
copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
© e Author(s) 2019
Content courtesy of Springer Nature, terms of use apply. Rights reserved

1.
2.
3.
4.
5.
6.
Terms and Conditions
Springer Nature journal content, brought to you courtesy of Springer Nature Customer Service Center GmbH (“Springer Nature”).
Springer Nature supports a reasonable amount of sharing of research papers by authors, subscribers and authorised users (“Users”), for small-
scale personal, non-commercial use provided that all copyright, trade and service marks and other proprietary notices are maintained. By
accessing, sharing, receiving or otherwise using the Springer Nature journal content you agree to these terms of use (“Terms”). For these
purposes, Springer Nature considers academic use (by researchers and students) to be non-commercial.
These Terms are supplementary and will apply in addition to any applicable website terms and conditions, a relevant site licence or a personal
subscription. These Terms will prevail over any conflict or ambiguity with regards to the relevant terms, a site licence or a personal subscription
(to the extent of the conflict or ambiguity only). For Creative Commons-licensed articles, the terms of the Creative Commons license used will
apply.
We collect and use personal data to provide access to the Springer Nature journal content. We may also use these personal data internally within
ResearchGate and Springer Nature and as agreed share it, in an anonymised way, for purposes of tracking, analysis and reporting. We will not
otherwise disclose your personal data outside the ResearchGate or the Springer Nature group of companies unless we have your permission as
detailed in the Privacy Policy.
While Users may use the Springer Nature journal content for small scale, personal non-commercial use, it is important to note that Users may
not:
use such content for the purpose of providing other users with access on a regular or large scale basis or as a means to circumvent access
control;
use such content where to do so would be considered a criminal or statutory offence in any jurisdiction, or gives rise to civil liability, or is
otherwise unlawful;
falsely or misleadingly imply or suggest endorsement, approval , sponsorship, or association unless explicitly agreed to by Springer Nature in
writing;
use bots or other automated methods to access the content or redirect messages
override any security feature or exclusionary protocol; or
share the content in order to create substitute for Springer Nature products or services or a systematic database of Springer Nature journal
content.
In line with the restriction against commercial use, Springer Nature does not permit the creation of a product or service that creates revenue,
royalties, rent or income from our content or its inclusion as part of a paid for service or for other commercial gain. Springer Nature journal
content cannot be used for inter-library loans and librarians may not upload Springer Nature journal content on a large scale into their, or any
other, institutional repository.
These terms of use are reviewed regularly and may be amended at any time. Springer Nature is not obligated to publish any information or
content on this website and may remove it or features or functionality at our sole discretion, at any time with or without notice. Springer Nature
may revoke this licence to you at any time and remove access to any copies of the Springer Nature journal content which have been saved.
To the fullest extent permitted by law, Springer Nature makes no warranties, representations or guarantees to Users, either express or implied
with respect to the Springer nature journal content and all parties disclaim and waive any implied warranties or warranties imposed by law,
including merchantability or fitness for any particular purpose.
Please note that these rights do not automatically extend to content, data or other material published by Springer Nature that may be licensed
from third parties.
If you would like to use or distribute our Springer Nature journal content to a wider audience or on a regular basis or in any other manner not
expressly permitted by these Terms, please contact Springer Nature at
onlineservice@springernature.com